Hematological stains and the Romanovsky effect

The development of the stains we use routinely today took place over long periods of time, with a lot of trial and error, mostly empirically, and thanks to the contributions of many researchers. Nowhere is this truer than in the case of hematological staining of blood elements.

Right from the start, eosin, an acid stain, was found to stain red blood cells with a pinkish hue, as well as the acidophilic granules of white blood cells, aptly named eosinophils. In a long quest to color malaria parasites which lasted many years, Dimitri Romanovsky came up with a solution. He discovered that by mixing an old methylene blue solution left on the lab bench with an acid stain,  he was getting what he was looking for. 

He unknowingly had achieved much more, for this mixture now included basic dyes, products of the oxidation of methylene blue - Azur A and Azur B, which stained basic structures such as basophilic leukocyte granules, neutrophil cytoplasm and nuclei. 

This happy mixture of eosin and oxidized methylene blue gives us panoptic staining with a single solution that differentially stains all blood elements. Aan additional mechanism known as metachromasia means that dye colors can be modified by the very structures of the colored components. This gives rise to a whole range of granulation and inclusion shades, which even today enable excellent differentiation for reliable diagnosis.

Numerous variations followed, developed by May Grunwald, Wright and Gustav Giemsa. But they all follow the same principle: a mixture of an acid dye such as eosin with a basic dye of varying proportions of azure A and B and methylene blue.

References

A brief history of dyes used in light microscopy : https://www.nirgal.net/microscopie/sub_colorants.html